Cytokinesis or cell division is the biological basis of all life. From bacteria to humans, the cytoskeleton plays an important role in cell division. In the bacterium, Escherichia coli, FtsZ - a homolog of the eukaryotic cytoskeletal protein tubulin, is the first known protein to localize to midcell, the site of future division. At least 19 essential and accessory proteins are involved in this process, all of which assemble into a large ring like multi- protein complex at midcell. The E. coli essential cell division machinery is well characterized, however, much remains to be discovered about the identities and role(s) of accessory factors in the maintenance and activity of the division complex. The proposed application focuses on characterizing the molecular interactions of 3 putative accessory division proteins identified by using a genome-wide sub-cellular protein localization library in E. coli and has 3 specific goals. One, to identify and characterize key domains and amino acid residues within the previously uncharacterized division proteins that allow localization to midcell using domain swaps, deletion assays, and random mutagenesis analyses. Non-localizing derivatives will be identified using a novel genetic screen. Two, to determine the recruitment pattern of these putative accessory proteins at midcell using depletion and premature targeting assays. Synergistic effects of accessory proteins on cell division will be examined by inactivating different combinations of genes. Three, to identify proteins that interact with the previously uncharacterized division proteins by examining known suppressors, and later extending genetic screens to identify additional factors. Interactions will be confirmed by two- hybrid and co-immunoprecipitation assays. The putative cell division accessory factors identified in this study in E. coli are highly conserved in a number of Gram-negatives, including pathogens, such as Shigella, Salmonella, Yersinia, and Vibrio. Molecular characterization of how the various factors influence FtsZ ring stability and activity at midcell could be exploited to identify a new generation of antimicrobial drug targets. Furthermore, our study will illuminate how accessory factors mediate spatiotemporal regulation of the FtsZ ring during cytokinesis, as also have important implications towards the general understanding of how localization of bacterial proteins is achieved. Using Escherichia coli as a model system, this study addresses a fundamental scientific problem of how bacterial cells divide at midcell. The important role of accessory proteins in regulating the stability and activity of the midcell division complex will be examined. Identification and characterization of the molecular mechanisms of how conserved accessory proteins govern division complex integrity could be exploited as targets in the development of new antibacterials.